A successful infection involves virus entry into the cell; uncoating, expression, and replication of the genome; assembly and release of infectious virus particles; and defense against specific and non-specific host immune mechanisms. Combined genetic, biochemical, electron microscopic, and immunologic approaches are being used to investigate these complex processes. During the past year we continued work on the assembly of vaccinia virus. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes. To study the role of the A17L protein in morphogenesis, we constructed inducer-dependent recombinant viruses in which the A17L ORF was under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis. With the aid of monoclonal antibodies, a 23- to 28-kDa glycoprotein that specifically localized to the outer envelope of vaccinia virus was shown to be encoded by the A33R gene. The amino acid sequence and hydrophilicity plot predicted that the A33R gene product is a type II membrane protein with two potential asparagine-linked glycosylation sites. Triton X-114 partitioning verified that the A33R product is an integral membrane protein and electron microscopy revealed that it is expressed on the surface of extracelluar enveloped virions. The functional relationships between two non-homologous poxvirus host range genes, K1L and CP77 were investigated. Recombinant vaccinia virus that had the K1L gene replaced by the CP77 gene were able to replicate in rabbit kidney cells suggesting that the viral proteins act in a common virus/cell interaction pathway.